Infection by flaviviruses such as dengue, yellow fever, and West Nile is a major medical and socio-economic problem worldwide, yet effective antiviral therapeutics to treat flavivirus infection are not currently available. As such, it is imperative that potent, selective, and cost-effective antiviral compounds be identified. The overall goal of this project is the discovery of novel inhibitors of the guanylyltransferase (GTase) active site of the flavivirus RNA capping enzyme (CE) that could serve as effective broad-spectrum chemotherapeutic agents for the treatment of flavivirus infection. The CE generates the cap structure at the 5' end of viral RNAs that is required for efficient translation of the viral genome and is essential for viral growth. The GTase transfers a guanosine monophosphate from GTP to the 5' end of the genomic RNA to produce the cap 0 structure. Disruption of GTP binding by the CE inhibits viral replication, indicating that this function is critical to viral viability. We recently published that the GTP binding site harbors the GTase active site, increasing interest in the GTP-binding site as a potential drug target site. We have designed and successfully implemented a simple and rapid in vitro high-throughput assay to identify compounds that interfere with GTP binding by the CE. In this application we propose to expand on our previous screening by performing additional HTS within the MLPCN with the goal of identifying and optimizing a chemically diverse set of CE GTP-binding inhibitors in order to identify lead compounds for drug development. The best inhibitors (in terms of breadth, potency and drug-like characteristics) will be tested for their ability to inhibit GTase enzymatic activity, cytotoxicity, and antiviral activity in cell culture. This project will result in the rapid and efficient identification of inhibitors of flavivirus replication with the ultimate goal of describing lead compounds with drug-like properties suitable for preclinical development for the treatment flavivirus infection. PUBLIC HEALTH RELEVANCE: Disease resulting from mosquito-borne flavivirus infection causes significant human morbidity and mortality throughout the world, yet there currently are no effective therapeutics to treat infected patients. The goal of this project is to identify and characterize broadly active antiviral compounds targeting the guanylyltransferase active site of the conserved flavivirus capping enzyme.